Macrophages are myeloid lineage cells that reside in virtually all tissues of the body and play essential roles in immunity, tissue homeostasis and wound repair. Responses to bacterial and viral infection are associated with a 'classical' program of macrophage activation involving NF?B and other signal-dependent transcription factors that direct expression of genes that promote inflammation and initiate innate and adaptive immune responses. Low-grade forms of classical macrophage activation are causally linked to the pathogenesis of numerous inflammatory diseases that include type 2 diabetes. Conversely, resident macrophages in healthy adipose tissue exhibit features of an 'alternative' program of macrophage activation, involving the IL4/ STAT6/PPAR??axis. The STAT6/PPAR?-dependent transcriptional program is linked to expression of genes that actively maintain tissue homeostasis and insulin sensitivity. Although the pathways responsible for classical and alternative activation are highly conserved, individual variation in the functional responses of these pathways is proposed to influence risk for diseases in which inflammation plays a pathogenic role. In this application, we propose to build on recent conceptual and technical advances and the vast natural genetic variation provided by inbred strains of mice to comprehensively decode the transcriptional circuitry underlying classical and alternative macrophage activation. These studies will test and extend a general collaborative/hierarchical model for the selection and function of signal-dependent enhancers required for the transcriptional outputs of NF?B and PPAR??through three Specific Aims. Specific Aim 1 will leverage the >50 million SNPs provided by 5 inbred strains of mice to identify the collaborating transcription factors required for the genome-wide binding patterns of NF?B and PPAR??in classically or alternatively activated macrophages, respectively. Specific Aim 2 will extend these studies to define the requirements for the transition from transcription factor binding to enhancer activation. This aim is based on the hypothesis that an essential feature of a functional enhancer is that it is actively transcribed. Specific Aim 3 will use a novel form of network modeling informed by effects of natural genetic variation and chromatin connectivity maps to functionally link specific enhancers to target genes. General predictions regarding the modularity of NF?B and PPAR?-activated enhancers will be tested in human macrophages. If successful, these studies will result in qualitative advances in our understanding of classical and alternative macrophage activation, enable insights into effects of natural genetic variation on these responses and disease, and provide a general approach for decoding the transcriptional regulatory circuitry for any signal-dependent program of gene expression in any cell type.